Standard Operating Procedure for Frozen Tissue Section Preparation and Section Storage
Standard Operating Procedure for Frozen Tissue Section Preparation and Section Storage
1 Overview
1.1 Purpose and Scope
This procedure is intended to standardize tissue pretreatment, fixation, sucrose cryoprotection, OCT embedding, rapid freezing, cryosectioning, section collection, frozen storage, and thawing procedures. It is applicable to immunofluorescence, immunohistochemistry, enzyme histochemistry, lipid staining, and routine histomorphological studies of animal tissues and research tissue samples.
1.2 Principle
After rapid freezing, water within the tissue solidifies, providing sufficient hardness for sectioning. The tissue can then be cut into sections 5-20 μm thick using a cryostat. OCT cryoembedding medium fixes the orientation of the tissue, fills the spaces surrounding the tissue, and provides mechanical support at low temperatures.
After fixation, sufficient infiltration with sucrose solution reduces the amount of free water in the tissue and decreases cellular and tissue structural damage caused by the formation of large ice crystals during freezing. After sectioning, tissue sections may be directly mounted onto glass slides or collected in frozen section storage solution and stored at -20°C. Before subsequent experiments, stored sections can be thawed and washed.
2 Materials and Reagents
2.1 Tissue Samples
(1) Fresh animal tissues.
(2) Perfusion-fixed animal tissues.
(3) Surgically resected or biopsy tissues.
Tissues should be processed as soon as possible after collection to prevent drying, autolysis, or prolonged exposure to room temperature.
2.2 Main Reagents
(1) PBS, pH 7.4.
(2) 4% paraformaldehyde fixative.
(3) 15% sucrose solution.
(4) 30% sucrose solution.
(5) OCT cryoembedding medium.
(6) Frozen section storage solution.
(7) Liquid nitrogen.
(8) Isopentane.
(9) Histological-grade acetone.
(10) Methanol.
2.3 Main Consumables
(1) Tissue embedding molds.
(2) Cryostat specimen chucks.
(3) Disposable microtome blades.
(4) Adhesive slides, positively charged slides, or gelatin-coated slides.
(5) 24-well or 48-well plates.
(6) Fine brushes or sectioning brushes.
(7) Forceps.
(8) Absorbent paper.
(9) Sample storage boxes.
2.4 Main Equipment
(1) Cryostat.
(2) -80°C freezer.
(3) -20°C freezer.
(4) 4°C refrigerator.
(5) Horizontal shaker.
(6) Liquid nitrogen tank.
(7) Fume hood.
3 Reagent Preparation
3.1 15% Sucrose Solution
Weigh 15 g of sucrose and add approximately 80 mL of PBS. After complete dissolution, bring the final volume to 100 mL with PBS. Sterilize by filtration and store at 4°C.
3.2 30% Sucrose Solution
Weigh 30 g of sucrose and add approximately 70 mL of PBS. After complete dissolution, bring the final volume to 100 mL with PBS. Sterilize by filtration and store at 4°C.
Sucrose solutions should remain clear. Do not continue using a solution if turbidity, precipitation, or microbial contamination is observed.
4 Tissue Pretreatment
4.1 Tissue Collection and Trimming
(1) Immediately place the collected tissue in prechilled PBS.
(2) Gently wash the tissue surface with PBS to remove blood and other residues.
(3) Remove irrelevant adipose tissue, connective tissue, and necrotic tissue.
(4) Trim the tissue to a size suitable for embedding. Tissue thickness should generally not exceed 5-10 mm.
(5) Determine the intended plane of observation and record the tissue orientation.
If the tissue block is too large, fixation, sucrose infiltration, and freezing of the central region will proceed slowly, increasing the risk of uneven fixation, ice crystal formation, or inconsistent tissue hardness.
4.2 Freezing of Fixed Tissues
(1) Tissue Fixation
Completely immerse the trimmed tissue in 4% paraformaldehyde fixative and fix on a shaker at 4°C for 2-4 h.
The fixation time should be adjusted according to the tissue type and tissue-block size. Larger or denser tissues may require a longer fixation time, but fixation for more than 24 h is generally not recommended because excessive cross-linking may interfere with antigen recognition.
(2) Tissue Washing
After fixation, wash the tissue three times with prechilled PBS for 5-10 min each.
(3) Sucrose Cryoprotection
Place the tissue in 15% sucrose solution at 4°C until it has substantially sunk.
After the tissue has substantially sunk, transfer it to 30% sucrose solution and continue incubation at 4°C until it has completely sunk.
Completion of sucrose cryoprotection should be determined primarily by tissue sinking rather than by a fixed incubation time alone.
4.3 Direct Freezing of Fresh Tissues
(1) Rapidly wash the tissue surface with prechilled PBS.
(2) Gently blot away excess liquid from the tissue surface using absorbent paper.
(3) Do not compress the tissue or leave it exposed to room temperature for an extended period.
(4) Immediately proceed with OCT embedding and rapid freezing.
After direct freezing of fresh tissue, post-section fixation may be performed using 4% paraformaldehyde, cold acetone, or cold methanol according to the requirements of the subsequent assay.
5 OCT Embedding and Rapid Tissue Freezing
5.1 Tissue Embedding
(1) Label the tissue embedding mold with the sample number, tissue type, and intended plane of observation.
(2) Add a small amount of prechilled OCT cryoembedding medium to the bottom of the embedding mold.
(3) Use forceps to place the tissue into the OCT, with the intended plane of observation facing the bottom of the mold.
(4) Adjust the position and orientation of the tissue so that it remains flat.
(5) Continue adding OCT until the tissue is completely covered.
(6) Check for visible air bubbles around the tissue and gently remove them with a needle or forceps when necessary.
5.2 Prechilling of Isopentane
(1) Add an appropriate volume of isopentane to a cryogenic-resistant container.
(2) Place the container of isopentane in liquid nitrogen for external cooling.
(3) Use the isopentane after it has cooled sufficiently and begins to appear slightly viscous or develops crystals around the edges.
(4) Do not allow the isopentane to freeze completely.
5.3 Rapid Tissue Freezing
(1) Place the embedding mold containing the tissue and OCT into the prechilled isopentane.
(2) Keep the embedding mold stable to prevent displacement of the tissue.
(3) Remove the mold immediately after the OCT has completely turned white and solidified.
(4) Transfer the frozen tissue block to dry ice or a prechilled storage box.
(5) Do not leave the frozen tissue block at room temperature for an extended period.
Direct immersion of an unsealed tissue block in liquid nitrogen is not recommended because vigorous boiling of liquid nitrogen may cause OCT cracking, tissue displacement, or uneven freezing.
5.4 Storage of Embedded Tissue Blocks
(1) Confirm that the tissue block is completely frozen.
(2) Seal the tissue block in suitable packaging.
(3) Label the sample name, tissue type, treatment method, and storage date.
(4) Store at -80°C.
(5) Avoid repeated freeze-thaw cycles.
6 Cryosectioning
6.1 Cryostat Precooling
(1) Turn on the cryostat in advance.
(2) Initially set the chamber temperature to -18°C to -22°C.
(3) Begin sectioning only after the chamber, blade holder, specimen head, and rapid-freezing shelf have been sufficiently precooled.
(4) Clean the specimen stage, blade holder, anti-roll plate, and cryostat chamber.
Tissues with a high lipid content generally require a somewhat lower sectioning temperature. Tissues with a high water content or brittle texture may require a somewhat higher sectioning temperature.
6.2 Preparation of the Specimen Chuck
(1) Add a layer of OCT approximately 2-3 mm thick to the surface of the prechilled specimen chuck.
(2) Place the specimen chuck on the rapid-freezing shelf of the cryostat and allow the OCT to partially solidify.
(3) Remove the embedded tissue block from the -80°C freezer and remove the embedding mold.
(4) Place the embedded tissue block on the OCT surface of the specimen chuck according to the predetermined sectioning orientation.
(5) Add a small amount of OCT around the tissue block to secure it.
(6) After the OCT has completely solidified, install the specimen chuck onto the specimen head.
6.3 Tissue Temperature Equilibration
Allow the mounted tissue block to equilibrate in the cryostat for 20-30 min so that its temperature approaches that of the cryostat chamber.
If the tissue temperature is too low, sections may curl, fracture, or develop knife marks. If the tissue temperature is too high, sections may deform, adhere to the blade, or fail to maintain an intact morphology.
6.4 Blade Installation
(1) Lock the cryostat handwheel.
(2) Install a disposable microtome blade in the blade holder and secure it.
(3) Initially set the blade clearance angle to 5°-7°.
(4) Adjust the anti-roll plate so that it is close to, but not in direct contact with, the blade edge.
(5) Confirm that the blade holder, specimen chuck, and specimen head are firmly secured.
6.5 Tissue Trimming
(1) Set the section thickness to 20-50 μm.
(2) Slowly advance the tissue block for coarse trimming.
(3) Continue trimming until a complete tissue surface is exposed.
(4) Examine the tissue for internal cracks, cavities, obvious ice crystals, or incompletely frozen regions.
(5) Adjust the tissue orientation or sectioning temperature when necessary.
(6) After a complete tissue surface has been obtained, adjust the thickness to the desired section thickness.
6.6 Final Sectioning
Select an appropriate section thickness according to the subsequent experiment:
(1) Routine immunofluorescence: 5-10 μm.
(2) General histomorphological observation: 8-12 μm.
(3) Observation of thicker tissue structures or free-floating sections: 10-20 μm.
Rotate the handwheel at a stable and uniform speed. If the section curls, use a fine brush or sectioning brush to gently guide the leading edge so that the section spreads flat over the blade holder or anti-roll plate.
7 Section Collection and Storage
7.1 Slide-Mounting Method
(1) Bring an adhesive slide close to the flattened tissue section.
(2) Use the temperature difference between the slide and the frozen section to allow the section to adhere naturally to the slide surface.
(3) Alternatively, use a fine brush to gently transfer the flattened section onto the slide.
(4) Avoid repeatedly rubbing the tissue area with the brush.
(5) Maintain appropriate spacing among multiple tissue sections on the same slide.
(6) Label the edge of the slide with the sample number and section sequence.
(7) Place the slide horizontally and allow it to warm and dry at room temperature for 20-30 min.
(8) Sections intended for experiments within a short period may be fixed or stained directly.
(9) For long-term storage, seal completely dried sections and store them at -80°C.
7.2 Frozen Storage of Free-Floating Sections
(1) Prepare a 24-well or 48-well plate according to the number of sections.
(2) Add 1-2 mL of frozen section storage solution to each well.
(3) Use a prechilled fine brush or sectioning brush to gently transfer the tissue sections into the wells.
(4) Confirm that the sections are completely immersed in the storage solution and are not folded or attached to the well wall.
(5) Collect the sections sequentially according to the sample number and section order.
(6) Seal the plate and label it with the sample information, section numbers, and storage date.
(7) Store the plate at -20°C.
8 Thawing and Washing of Stored Sections
8.1 Section Thawing
(1) Before the experiment, remove the plate containing the sections from -20°C.
(2) Place the plate at room temperature for approximately 10 min.
(3) Proceed with subsequent operations after the storage solution has returned to a fluid state.
(4) Do not use a high-temperature water bath or heating plate to accelerate thawing.
8.2 Removal of Storage Solution
(1) After confirming the position of the section, slowly aspirate the storage solution from the side opposite the section.
(2) Add PBS prechilled to 4°C until the section is completely immersed.
(3) Gently wash on a shaker for 10 min.
(4) Remove the PBS and wash twice with room-temperature PBS for 5 min each.
(5) After washing, proceed with fixation, blocking, permeabilization, antibody incubation, or histological staining.
Sections should remain moist throughout thawing, aspiration, and washing.
9 Post-Section Fixation
9.1 Fixation With 4% Paraformaldehyde
(1) Wash the sections with PBS.
(2) Add 4% paraformaldehyde fixative until the sections are completely covered.
(3) Fix at room temperature for 10-15 min.
(4) Wash three times with PBS for 5 min each after fixation.
This method is suitable for most routine immunofluorescence and histomorphological experiments.
9.2 Cold Acetone Fixation
(1) Prechill histological-grade acetone to -20°C.
(2) Completely immerse the sections in cold acetone.
(3) Fix at -20°C for 10 min.
(4) Remove the sections and briefly air-dry them in a fume hood.
(5) Wash three times with PBS for 5 min each.
9.3 Cold Methanol Fixation
(1) Prechill methanol to -20°C.
(2) Completely immerse the sections in cold methanol.
(3) Fix at -20°C for 5-10 min.
(4) Remove the sections and wash three times with PBS for 5 min each.
Cold acetone and cold methanol fixation conditions should be confirmed through preliminary experiments according to the target antigen and subsequent staining method.
10 Common Problems and Cause Analysis
10.1 Section Curling
(1) The tissue block temperature is too low.
(2) The anti-roll plate is improperly positioned.
(3) The sectioning speed is too high.
(4) The blade clearance angle is inappropriate.
The sectioning temperature may be increased moderately, and the positions of the anti-roll plate and blade angle should be readjusted.
10.2 Sections Adhering to the Blade
(1) The tissue block temperature is too high.
(2) The tissue or OCT is not completely frozen.
(3) Moisture or contamination is present on the blade surface.
(4) The cryostat chamber temperature fluctuates substantially.
The sectioning temperature may be decreased moderately, the tissue equilibration time may be extended, and the blade should be cleaned or replaced.
10.3 Section Fragmentation or Cracking
(1) The tissue temperature is too low.
(2) The tissue is overfixed.
(3) Sucrose cryoprotection is insufficient.
(4) The tissue freezing rate is too slow.
(5) The blade is dull or nicked.
(6) The sectioning speed is unstable.
10.4 Honeycomb-Like Vacuoles in the Tissue
(1) The tissue freezing rate is too slow.
(2) The tissue block is too large.
(3) The fixed tissue has not undergone sufficient sucrose cryoprotection.
(4) Large ice crystals have formed within the tissue.
10.5 Uneven Section Thickness
(1) The specimen chuck, blade holder, or specimen head is not securely locked.
(2) The tissue surface is not parallel to the blade.
(3) The cryostat has mechanical vibration.
(4) The tissue block has uneven hardness.
(5) The handwheel rotation speed is unstable.
10.6 Tissue Detachment From the Slide
(1) An untreated standard slide was used.
(2) The section was not allowed to adhere and dry sufficiently.
(3) Tissue fixation was insufficient.
(4) Subsequent washing was too vigorous.
(5) The tissue section was too thick.
10.7 Folding or Wall Adhesion of Stored Sections
(1) The section was not completely flattened.
(2) The brush directly compressed the section.
(3) The volume of storage solution was insufficient.
(4) The section was not completely immersed.
The section should be gently transferred only after it has been completely flattened, and the storage solution should fully cover the section.
10.8 High Background in Subsequent Staining
(1) The storage solution was not sufficiently washed away.
(2) Antibody incubation was performed immediately after thawing.
(3) PBS washing time was insufficient.
(4) The section dried during processing.
The sections should be thoroughly washed with prechilled PBS and room-temperature PBS as specified before blocking, permeabilization, or antibody incubation.
11 Safety and Waste Disposal
(1) Wear cryogenic gloves and protective goggles when handling liquid nitrogen and low-temperature samples.
(2) Do not place sealed containers directly into liquid nitrogen.
(3) Isopentane is highly flammable and should be kept away from ignition sources and handled in a well-ventilated area.
(4) Acetone and methanol are flammable and volatile and should be handled in a fume hood.
(5) Paraformaldehyde is irritating and potentially toxic and should be handled in a fume hood.
(6) Before installing, adjusting, or removing the microtome blade, lock the cryostat handwheel and use the blade guard.
(7) Discard used blades in a designated sharps container.
(8) Tissue waste, fixatives, storage solution, and organic solvent waste should be collected separately and disposed of in accordance with laboratory biosafety and chemical waste management requirements.
12 Products Related to Frozen Tissue Section Preparation and Storage
Catalog # | Product Name | Grade & Purity | Corresponding Step | Application |
PBS (pH 7.4, Sterile) | Sterile-filtered, BioReagent, Low Endotoxin, for Cell culture | Tissue washing, washing after fixation, and section washing | Preferred for tissue processing and section washing | |
PBS, DNase&RNase Free | Sterile-filtered, BioReagent, DNase, RNase free, Ready-to-use, for Cell culture, 1× | Tissue and section washing | Suitable for experiments involving subsequent nucleic acid analysis | |
PhosphateBuffered Saline（PBS）1X concentrate | 1X,sterile,pH7.2-7.4 | Tissue and section washing | Used as ready-to-use PBS | |
PhosphateBuffered Saline（PBS）20X concentrate | sterile | PBS preparation | Diluted to 1× for tissue and section washing | |
PBS | 1 L/pouch | PBS preparation | Used for batch preparation of tissue-washing and section-washing buffer | |
Paraformaldehyde Fix Solution | 4% in PBS | Tissue prefixation and post-section fixation | Used for tissue fixation or frozen-section fixation | |
Sucrose-Paraformaldehyde Fix Solution (5%) | Suitable for Immunofluorescence(IF), BioReagent, Ready-to-use, Suitable for Immunohistochemistry(IHC), 5% | Tissue fixation | Used for selected fixed-tissue cryoprocessing procedures | |
Sucrose-Paraformaldehyde Fix Solution (10%) | Suitable for Immunofluorescence(IF), BioReagent, Ready-to-use, Suitable for Immunohistochemistry(IHC), 10% | Tissue fixation | Used in sucrose-containing tissue-fixation systems | |
Sucrose-Paraformaldehyde Fix Solution (20%) | Suitable for Immunofluorescence(IF), BioReagent, Ready-to-use, Suitable for Immunohistochemistry(IHC), 20% | Tissue fixation | Used for fixation and prefreezing treatment of selected tissues | |
Sucrose-Paraformaldehyde Fix Solution (30%) | Suitable for Immunofluorescence(IF), BioReagent, Ready-to-use, Suitable for Immunohistochemistry(IHC), 30% | Tissue fixation | Used in high-sucrose-concentration tissue-fixation systems | |
Sucrose | Moligand™, Ultra pure, ≥99.9%, RNase,DNase Free | Preparation of sucrose cryoprotective solutions | Preferred for preparation of 15% and 30% sucrose solutions | |
Sucrose | Moligand™, for Cell culture, Suitable for insect cell culture, ≥99.5% | Preparation of sucrose cryoprotective solutions | Used to prepare tissue cryoprotective solutions | |
Sucrose | Moligand™, Suitable for molecular biology, ≥99.5%(HPLC) | Preparation of sucrose cryoprotective solutions | Suitable for samples intended for subsequent molecular analysis | |
Frozen Section Embedding Medium | BioReagent, Suitable for Immunohistochemistry(IHC) | Tissue embedding and fixation to the specimen chuck | Preferred for tissue cryoembedding and cryosectioning | |
Frozen section Medium |
| Tissue cryoembedding | Used as an embedding medium for frozen sectioning | |
Methanol | AR, ≥99.5% | Post-section fixation | Used for antigen-detection procedures suitable for cold methanol fixation | |
Tissue-Freezing Medium | BioReagent | Section collection, frozen storage, and preservation | Used to preserve frozen sections at -20°C |
For more related articles, please see below:
[1] Immunofluorescence of frozen sections
[2] Frozen section fixation experiment
[3] In situ hybridization experiments on frozen sections
[4] Fluorescent staining of frozen sections for amyloid (AMYLOID) thioflavine T